Salmonella enterica serovar Typhimurium has a very broad host range and can colonize different Phyla. To date, only a small fraction of its genome has been surveyed for genes that differ in their contribution to colonization of different hosts. This project is designed to determine which Typhimurium mutants are unable to fully compete with wild type during colonization of three vertebrate host species, selected for their phylogenetic diversity and known susceptibility to Typhimurium infection: mouse, pig, and chicken. A pool of specific knockout mutants in every non-essential Typhimurium gene in vitro (~3900 knockouts) will be delivered intragastrically to the model animals. Short-term colonization in the gut will be monitored by dissection of host tissues at three days post infection. An oligonucleotide array with a novel design will be used to identify all mutants that become rarer during competitive colonization, indicating a role of that gene in the process. A novel fluctuation test will assure that only appropriate experiments will be applied to arrays. We have confirmed that the proposed assay detects almost all genes that are known to contribute to intraperitoneal and intragastric infection in the mouse. If the data suggest that a known colonization mechanism in one host may not be used in other hosts, this will be independently confirmed by testing single exemplar gene knockout mutants in these species. Novel host-restricted colonization mechanisms are beyond the scope of this two year project and will be studied, only if time and resources permit.